Expression system

ABSTRACT

An immunogenic reagent which produces an immune response which is protective against  Bacillus anthracis,  said reagent comprising one or more polypeptides which together represent up to three domains of the full length Protective Antigen (PA) of  B. anthracis  or variants of these, and at least one of said domains comprises domain 1 or domain 4 of PA or a variant thereof. The polypeptides of the immunogenic reagent as well as full length PA are produced by expression from  E. coli.  High yields of polypeptide are obtained using this method. Cells, vectors and nucleic acids used in the method are also described and claimed.

[0001] The present invention relates to polypeptides which produce animmune response which is protective against infection by Bacillusanthracis, to methods of producing these, to recombinant Escherichiacoli cells, useful in the methods, and to nucleic acids andtransformation vectors used.

[0002] Present systems for expressing Protective Antigen (PA) forvaccine systems use protease deficient Bacillus subtilis as theexpression host. Although such systems are acceptable in terms ofproduct quantity and purity, there are significant drawbacks. Firstly,regulatory authorities are generally unfamiliar with this host, andlicensing decisions may be delayed as a result. More importantly, thecurrently used strains of Bacillus subtilis produce thermostable sporeswhich require the use of a dedicated production plant.

[0003] WO00/02522 describes in particular VEE virus replicons whichexpress PA or certain immunogenic fragments.

[0004]E. coli is well known as an expression system for a range of humanvaccines. While the ability to readily ferment E. coli to very highcellular densities makes this bacterium an ideal host for the expressionof many proteins, previous attempts to express and purify recombinant PAfrom E. coli cytosol have been hindered by low protein yields andproteolytic degradation (Singh et al., J. Biol. Chem. (1989) 264;11099-11102, Vodkin et al., Cell (1993) 34; 693-697 and Sharma et al.,Protein Expr. purif. (1996), 7, 33-38).

[0005] A strategy for overexpressing PA as a stable, soluble protein inthe E. coli cytosol has been described recently (Willhite et al.,Protein and Peptide Letters, (1998), 5; 273-278). The strategy adoptedis one of adding an affinity tag sequence to the N terminus of PA, whichallows a simple purification system. A problem with this system is thatit requires a further downstream processing step in order to remove thetag before the PA can be used.

[0006] Codon optimisation is a technique which is now well known andused in the design of synthetic genes. There is a degree of redundancyin the genetic code, in so far as most amino acids are coded for by morethan one codon sequence. Different organisms utilise one or other ofthese different codons preferentially. By optimising codons, it isgenerally expected that expression levels of the particular protein willbe enhanced.

[0007] This is generally desirable, except where, as in the case of PA,higher expression levels will result in proteolytic degradation and/orcell toxicity. In such cases, elevating expression levels might becounter-productive and result in significant cell toxicity.

[0008] Surprisingly however, the applicants have found that this is notthe case in E. coli and that in this system, codon optimisation resultsin expression of unexpectedly high levels of recombinant PA,irrespective of the presence or absence of proteolytic enzymes withinthe strain.

[0009] Furthermore, it would appear that expression of a protectivedomain of PA does not inhibit expression in E. coli.

[0010] The crystal structure of native PA has been elucidated (PetosaC., et al. Nature 385: 833-838,1997) and shows that PA consists of fourdistinct and functionally independent domains: domain 1, divided into1a, 1˜167 amino acids and 1b, 168-258 amino acids; domain 2, 259-487amino acids; domain 3, 488-595 amino acids and domain 4, 596-735 aminoacids.

[0011] The applicants have identified that certain domains appear toproduce surprisingly good protective effects when used in isolation, infusion proteins or in combination with each other.

[0012] According to the present invention there is provided animmunogenic reagent which produces an immune response which isprotective against Bacillus anthracis, said reagent comprising one ormore polypeptides which together represent up to three domains of thefull length Protective Antigen (PA) of B. anthracis or variants ofthese, and at least one of said domains comprises domain 1 or domain 4of PA or a variant thereof.

[0013] Specifically, the reagent will comprise mixtures of polypeptidesor fusion peptides wherein individual polypeptides comprise one of moreindividual domains of PA.

[0014] In particular, the reagent comprises polypeptide(s) comprisingdomain 1 or domain 4 of PA or a variant thereof, in a form other thanfull length PA. Where present, domains are suitably complete, inparticular domain 1 is present in its entirety.

[0015] The term “polypeptide” used herein includes proteins andpeptides.

[0016] As used herein, the expression “variant” refers to sequences ofamino acids which differ from the basic sequence in that one or moreamino acids within the sequence are deleted or substituted for otheramino acids, but which still produce an immune response which isprotective against Bacillus anthracis. Amino acid substitutions may beregarded as “conservative” where an amino acid is replaced with adifferent amino acid with broadly similar properties. Non-conservativesubstitutions are where amino acids are replaced with amino acids of adifferent type. Broadly speaking, fewer non-conservative substitutionswill be possible without altering the biological activity of thepolypeptide. Suitably variants will be at least 60% identical,preferably at least 75% identical, and more preferably at least 90%identical to the PA sequence.

[0017] In particular, the identity of a particular variant sequence tothe PA sequence may be assessed using the multiple alignment methoddescribed by Lipman and Pearson, (Lipman, D. J. & Pearson, W. R. (1985)Rapid and Sensitive Protein Similarity Searches, Science, vol 227,pp1435-1441). The “optimised” percentage score should be calculated withthe following parameters for the Lipman-Pearson algorithm:ktup=1, gappenalty=4 and gap penalty length=12. The sequences for which similarityis to be assessed should be used as the “test sequence” which means thatthe base sequence for the comparison, (SEQ ID NO 1), should be enteredfirst into the algorithm.

[0018] Preferably, the reagent of the invention includes a polypeptidewhich has the sequence of domain 1 and/or domain 4 of wild-type PA.

[0019] A particularly preferred embodiment of the invention comprisesdomain 4 of the PA of B. anthracis.

[0020] These domains comprise the following sequences shown in thefollowing Table 1. TABLE 1 Domain Amino acids of full-length PA* 4596-735 1  1-258

[0021] These amino acid numbers refer to the sequence as shown in Welkoset al. Gene 69 (1988) 287-300 and are illustrated hereinafter as SEQ IDNOs 15 (FIG. 4) and 3 (FIG. 3) respectively.

[0022] Domain 1 comprises two regions, designated 1a and 1b. Region 1acomprises amino acids 1-167 whereas region 1b is from amino acid168-258. It appears that region 1a is important for the production of agood protective response, and the full domain may be preferred.

[0023] In a particularly preferred embodiment, a combination of domains1 and 4 or protective regions thereof, are used as the immunogenicreagent which gives rise to an immune response protective against B.anthracis. This combination, for example as a fusion peptide, may beexpressed using the expression system of the invention as outlinedhereinafter.

[0024] When domain 1 is employed, it is suitably fused to domain 2 ofthe PA sequence, and may preferably be fused to domain 2 and domain 3.

[0025] Such combinations and their use in prophylaxis or therapy forms afurther aspect of the invention.

[0026] Suitably the domains described above are part of a fusionprotein, preferably with an N-terminal glutathione-s-transferase protein(GST). The GST not only assists in the purification of the protein, itmay also provide an adjuvant effect, possibly as a result of increasingthe size.

[0027] The polypeptides of the invention are suitably prepared byconventional methods. For example, they may be synthesised or they maybe prepared using recombinant DNA technology. In particular, nucleicacids which encode said domains are included in an expression vector,which is used to transform a host cell. Culture of the host cellfollowed by isolation of the desired polypeptide can then be carried outusing conventional methods. Nucleic acids, vectors and transformed cellsused in these methods form a further aspect of the invention.

[0028] Generally speaking, the host cells used will be those that areconventionally used in the preparation of PA, such as Bacillus subtilis.

[0029] The applicants have found surprisingly that the domains either inisolation or in combination, may be successfully expressed in E. coliunder certain conditions.

[0030] Thus, the present invention further provides a method forproducing an immunogenic polypeptide which produces an immune responsewhich is protective against B. anthracis, said method comprisingtransforming an E. coli host with a nucleic acid which encodes either(a) the protective antigen (PA) of Bacillus anthracis or a variantthereof which can produce a protective immune response, or (b) apolypeptide comprising at least one protective domain of the protectiveantigen (PA) of Bacillus anthracis or a variant thereof which canproduce a protective immune response as described above, culturing thetransformed host and recovering the polypeptide therefrom, provided thatwhere the polypeptide is the protective antigen (PA) of Bacillusanthracis or a variant thereof which can produce a protective immuneresponse, the percentage of guanidine and cytosine residues within thesaid nucleic acid is in excess of 35%.

[0031] Using these options, high yields of product can be obtained usinga favoured expression host.

[0032] A table showing codons and the frequency with which they appearin the genomes of Escherichia coli and Bacillus anthracis is shown inFIG. 1. It is clear that guanidine and cytosine appear much morefrequently in E. coli than B. anthracis. Analysis of the codon usagecontent reveals the following: 1^(st) letter of 2nd letter 3rd letterTotal GC Species Codon GC of Codon GC of Codon GC content E. coli 58.50%40.70% 54.90% 51.37% B. anthracis 44.51% 31.07% 25.20% 33.59%

[0033] Thus it would appear that codons which are favoured by E. coliare those which include guanidine or cytosine where possible.

[0034] By increasing the percentage of guanidine and cytosinenucleotides in the sequence used to encode the immunogenic protein overthat normally found in the wild-type B. anthracis gene, the codon usagewill be such that expression in E. coli is improved.

[0035] Suitably the percentage of guanidine and cytosine residues withinthe coding nucleic acid used in the invention, at least where thepolypeptide is the protective antigen (PA) of Bacillus anthracis or avariant thereof which can produce a protective immune response, is inexcess of 40%, preferably in excess of 45% and most preferably from50-52%.

[0036] High levels of expression of protective domains can be achieved,with using the wild-type B. anthracis sequence encoding these units.However, the yields may be improved further by increasing the GC % ofthe nucleic acid as described above.

[0037] In a particular embodiment, the method involves the expression ofPA of B. anthracis.

[0038] Further according to the present invention, there is provided arecombinant Escherichia coli cell which has been transformed with anucleic acid which encodes the protective antigen (PA) of Bacillusanthracis or a variant thereof which can produce a protective immuneresponse, and wherein the percentage of guanidine and cytosine residueswithin the nucleic acid is in excess of 35%.

[0039] As before, suitably the percentage of guanidine and cytosineresidues within the coding nucleic acid is in excess of 40%, preferablyin excess of 45% and most preferably from 50-52%.

[0040] Suitably, the nucleic acid used to transform the E. coli cells ofthe invention is a synthetic gene. In particular, the nucleic acid is ofSEQ ID NO 1 as shown in FIG. 2 or a modified form thereof.

[0041] The expression “modified form” refers to other nucleic acidsequences which encode PA or fragments or variants thereof which producea protective immune response but which utilise some different codons,provided the requirement for the percentage GC content in accordancewith the invention is met. Suitable modified forms will be at least 80%similar, preferably 90% similar and most preferably at least 95% similarto SEQ ID NO 1. In particular, the nucleic acid comprises SEQ ID NO 1.

[0042] In an alternative embodiment, the invention provides arecombinant Escherichia coli cell which has been transformed with anucleic acid which encodes a protective domain of the protective antigen(PA) of Bacillus anthracis or a variant thereof which can produce aprotective immune response.

[0043] Preferably, the nucleic acid encodes domain 1 or domain 4 of B.anthracis.

[0044] Further according to the invention there is provided a method ofproducing an immunogenic polypeptide which produces an immune responsewhich is protective against B. anthracis, said method comprisingculturing a cell as described above and recovering the desiredpolypeptide from the culture. Such methods are well known in the art.

[0045] In yet a further aspect, the invention provides an E. colitransformation vector comprising a nucleic acid which encodes theprotective antigen (PA) of Bacillus anthracis or a variant thereof whichcan produce a protective immune response, and wherein the percentage ofguanidine and cytosine residues within the nucleic acid is in excess of35%.

[0046] A still further aspect of the invention comprises an E. colitransformation vector comprising a nucleic acid which encodes aprotective domain of the protective antigen (PA) of Bacillus anthracisor a variant thereof which can produce a protective immune response.

[0047] Suitable vectors for use in the transformation of E. coil arewell known in the art. For example, the T7 expression system providesgood expression levels. However a particularly preferred vectorcomprises pAG163 obtainable from Avecia (UK).

[0048] A nucleic acid of SEQ ID NO 1 or a variant thereof which encodesPA and which has at least 35%, preferably at least 40%, more preferablyat least 45% and most preferably from 50-52% GC content form a furtheraspect of the invention.

[0049] If desired, PA of the variants, or domains can be expressed as afusion to another protein, for example a protein which provides adifferent immunity, a protein which will assist in purification of theproduct or a highly expressed protein (e.g. thioredoxin, GST) to ensuregood initiation of translation.

[0050] Optionally, additional systems will be added such as T7 lysozymeto the expression system, to improve the repression of the system,although, in the case of the invention, the problems associated withcell toxicity have not been noted.

[0051] Any suitable E. coli strain can be employed in the process of theinvention. Strains which are deficient in a number of proteases (e.g.Ion⁻, ompT⁻) are available, which would be expected to minimiseproteolysis. However, the applicants have found that there is no need touse such strains to achieve good yields of product and that other knownstrains such as K12 produce surprisingly high product yields.

[0052] Fermentation of the strain is generally carried out underconventional conditions as would be understood in the art. For example,fermentations can be carried out as batch cultures, preferably in largeshake flasks, using a complex medium containing antibiotics for plasmidmaintenance and with addition of IPTG for induction.

[0053] Suitably cultures are harvested and cells stored at −20° C. untilrequired for purification.

[0054] Suitable purification schemes for E. coli PA (or variant ordomain) expression can be adapted from those used in B. subtilisexpression. The individual purification steps to be used will depend onthe physical characteristics of recombinant PA. Typically an ionexchange chromatography separation is carried out under conditions whichallow greatest differential binding to the column followed by collectionof fractions from a shallow gradient. In some cases, a singlechromatographic step may be sufficient to obtain product of the desiredspecification.

[0055] Fractions can be analysed for the presence of the product usingSDS PAGE or Western blotting as required.

[0056] As illustrated hereinafter, the successful cloning and expressionof a panel of fusion proteins representing intact or partial domains ofrPA has been achieved. The immunogenicity and protective efficacy ofthese fusion proteins against STI spore challenge has been assessed inthe A/J mouse model.

[0057] All the rPA domain proteins were immunogenic in A/J mice andconferred at least partial protection against challenge compared to theGST control immunised mice. The carrier protein, GST attached to theN-terminus of the domain proteins, did not impair the immunogenicity ofthe fusion proteins either in vivo, shown by the antibody responsestimulated in immunised animals, or in vitro as the fusion proteinscould be detected with anti-rPA antisera after Western blotting,indicating that the GST tag did not interfere with rPA epitoperecognition. Immunisation with the larger fusion proteins produced thehighest titres. In particular, mice immunised with the full length GST1-4 fusion protein produced a mean serum anti-rPA concentrationapproximately eight times that of the rPA immunised group (FIG. 5).Immunisation of mice with rPA domains 1-4 with the GST cleaved off,produced titres of approximately one half those produced by immunisationwith the fusion protein. Why this fusion protein should be much moreimmunogenic is unclear. It is possible that the increased size of thisprotein may have an adjuvantising effect on the immune effector cells.It did not stimulate this response to the same extent in the otherfusion proteins and any adjuvantising effect of the GST tag did notenhance protection against challenge as the cleaved proteins weresimilarly protective to their fusion protein counterparts.

[0058] Despite having good anti-rPA titres, some breakthrough inprotection at the lower challenge level of 10² MLD's, occurred in thegroups immunised with GST1, cleaved 1, GST1b-2, GST1b-3 and GST1-3 andimmunisation with these proteins did not prolong the survival time ofthose mice that did succumb to challenge, compared with the GST controlimmunised mice. This suggests that the immune response had not beenappropriately primed by these proteins to achieve full resistance to theinfection. As has been shown in other studies in mice and guinea pigs(Little S. F. et al. 1986. Infect. Immun. 52: 509-512, Turnbull P. C.B., et al., 1986. Infect. Immun. 52: 356-363) there is no precisecorrelation between antibody titre to PA and protection againstchallenge. However a certain threshold of antibody is required forprotection (Cohen S et. al., 2000 Infect. Immun. 68: 4549-4558),suggesting that cell mediated components of the immune response are alsorequired to be stimulated for protection (Williamson 1989).

[0059] GST1, GST1b-2 and GST1-2 were the least stable fusion proteinsproduced, as shown by SDS-Page and Western blotting results, possiblydue to the proteins being more susceptible to degradation in the absenceof domain 3, and this instability may have resulted in the loss ofprotective epitopes.

[0060] The structural conformation of the proteins may also be importantfor stimulating a protective immune response. The removal of Domain 1afrom the fusion proteins gave both reduced antibody titres and lessprotection against challenge, when compared to their intact counterpartsGST1-2 and GST1-3. Similarly, mice immunised with GST 1 alone werepartially protected against challenge, but when combined with domain 2,as the GST1-2 fusion protein, full protection was seen at the 10² MLDchallenge level. However the immune response stimulated by immunisationwith the GST1-2 fusion protein was insufficient to provide fullprotection against the higher 10³ MLD's challenge level, which againcould be due to the loss of protective epitopes due to degradation ofthe protein.

[0061] All groups immunised with truncates containing domain 4,including GST 4 alone, cleaved 4 alone and a mixture of two individuallyexpressed domains, GST 1 and GST 4 were fully protected againstchallenge with 10³ MLDs of STI spores (Table 1). Brossier et al showed adecrease in protection in mice immunised with a mutated strain of B.anthracis that expressed PA without domain 4 (Brossier F., et al. 2000.Infect. Immun. 68: 1781-1786) and this was confirmed in this study,where immunisation with GST 1-3 resulted in breakthrough in protectiondespite good antibody titres. These data indicate that domain 4 is theimmunodominant sub-unit of PA. Domain 4 represents the 139 amino acidsof the carboxy terminus of the PA polypeptide. It contains the host cellreceptor binding region (Little S. F. et al., 1996 Microbiology 142:707-715), identified as being in and near a small loop located betweenamino acid residues 679-693 (Varughese M., et al. 1999 Infect. Immun.67:1860-1865).

[0062] Therefore it is essential for host cell intoxication-as it hasbeen demonstrated that forms of PA expressed containing mutations(Varughese 1999 supra.) or deletions (Brossier 1999 supra.) in theregion of domain 4 are non-toxic. The crystal structure of PA showsdomain 4, and in particular a 19 amino acid loop of the domain(703-722), to be more exposed than the other three domains which areclosely associated with each other (Petosa 1997 supra.). This structuralarrangement may make domain 4 the most prominent epitope for recognitionby immune effector cells, and therefore fusion proteins containingdomain 4 would elicit the most protective immune response.

[0063] This investigation has further elucidated the role of PA in thestimulation of a protective immune response demonstrating thatprotection against anthrax infection can be attributed to individualdomains of PA.

[0064] The invention will now be particularly described by way ofexample, with reference to the accompanying drawings in which:

[0065]FIG. 1 is a Table of codon frequencies found within E. coli and B.anthracis;

[0066]FIG. 2 shows the sequence of a nucleic acid according to theinvention, which encodes PA of B. anthracis, as published by Welkos etal supra; and

[0067]FIG. 3 shows SEQ ID NOs 3-14, which are amino acid and DNAsequences used to encode various domains or combinations of domains ofPA as detailed hereinafter;

[0068]FIG. 4 shows SEQ ID NOs 15-16 which are the amino acid and DNAsequences of domain 4 of PA respectively; and

[0069]FIG. 5 is a table showing anti-rPA IgG concencentration, 37 dayspost primary immunisation, from A/J mice immunised intramuscularly ondays 1 and 28 with 10 g of fusion protein included PA fragment; resultsshown are mean±sem of samples taken from 5 mice per treatment group.

EXAMPLE 1

[0070] Investigation into Expression in E. coli

[0071] rPA expression plasmid pAG163::rPA has been modified tosubstitute Km^(R) marker for original Tc^(R) gene. This plasmid has beentransformed into expression host E. coli BLR (DE3) and expression leveland solubility assessed. This strain is deficient in the intracellularprotease La (Ion gene product) and the outer membrane protease OmpT.

[0072] Expression studies did not however show any improvement in theaccumulation of soluble protein in this strain compared to Ion+K12 hoststrains (i.e. accumulation is prevented due to excessive proteolysis).It was concluded that any intracellular proteolysis of rPA was not dueto the action of La protease.

EXAMPLE 2

[0073] Fermentation Analysis

[0074] Further analysis of the fermentation that was done using the K12strain UT5600 (DE3) pAG163::rPA.

[0075] It was found that the rPA in this culture was divided between thesoluble and insoluble fractions (estimated 350 mg/L insoluble, 650 mg/Lfull length soluble). The conditions used (37° C., 1 mM IPTG forinduction) had not yielded any detectable soluble rPA in shake flaskcultures and given the results described in Example 1 above, thepresence of a large amount of soluble rPA is surprising. Nevertheless itappears that manipulation of the fermentation, induction and point ofharvest may allow stable accumulation of rPA in E. coli K12 expressionstrains.

EXAMPLE 3

[0076] A sample of rPA was produced from material initially isolated asinsoluble inclusion bodies from the UT5600 (DE3) pAG163::rPAfermentation. Inclusion bodies were washed twice with 25 mM Tris-HCl pH8 and once with same buffer +2M urea. They were then solubilized inbuffer +8M urea and debris pelleted. Urea was removed by dilution into25 mM Tris-HCl pH 8 and static incubation overnight at 4° C. Dilutedsample was applied to Q sepharose column and protein eluted with NaClgradient. Fractions containing highest purity rPA were pooled, aliquotedand frozen at −70° C. Testing of this sample using 4-12% MES-SDS NuPAGEgel against a known standard indicated that it is high purity and low inendotoxin contamination.

EXAMPLE 4

[0077] Further Characterisation of the Product

[0078] N terminal sequencing of the product showed that the N-terminalsequence consisted of

[0079] MEVKQENRLL (SEQ ID NO 2)

[0080] This confirmed that the product was as expected with initiatormethionine left on.

[0081] The material was found to react in Western blot; MALDI MS on thesample indicated a mass of approx 82 700 (compared to expected mass of82 915). Given the high molecular mass and distance from mass standardused (66 KDa), this is considered an indication that material does nothave significant truncation but does not rule out microheterogeneitywithin the sample.

EXAMPLE 5

[0082] Testing of Individual Domains of PA

[0083] Individual domains of PA were produced as recombinant proteins inE. coli as fusion proteins with the carrier proteinglutathione-s-transferase (GST), using the Pharmacia pGEX-6P-3expression system. The sequences of the various domains and the DNAsequence used to encode them are attached herewith as FIG. 3. Therespective amino acid and DNA sequences are provided in Table 2 below.

[0084] These fusion proteins were used to immunise A/J mice (HarlanOlac) intramuscularly with 10 μg of the respective fusion proteinadsorbed to 20% v/v alhydrogel in a total volume of 100 μl.

[0085] Animals were immunised on two occasions and their development ofprotective immunity was determined by challenge with spores of B.anthracis (STI strain) at the indicated dose levels. The table belowshows survivors at 14 days post-challenge. Challenge level inspores/mouse Amino DNA acid SEQ SEQ ID Domains ID NO NO 5 × 10⁴ 9 × 10⁴9 × 10⁵ 1 × 10⁶ 5 × 10⁶ GST-1 3 4 4/4 3/5 GST-1 + 2 5 6 4/4; 4/5; 5/55/5 GST-1b + 2 7 8 2/5 1/5 GST-1b + 2 + 3 9 10 2/5 3/5 GST-1 + 2 + 3 1112 Nd 4/5 3/5 GST-1 + 2 + 3 + 4 13 14 Nd 5/5 5/5 1 + 2 + 3 + 4 13 14 NdNd 5/5 5/5

[0086] The data shows that a combination of all 4 domains of PA, whetherpresented as a fusion protein with GST or not, were protective up to ahigh challenge level. Removal of domain 4, leaving 1+2+3, resulted inbreakthrough at the highest challenge level tested, 9×10⁵. Domains 1+2were as protective as a combination of domains 1+2+3 at 9×10⁴ spores.However, removal of domain 1a to leave a GST fusion with domains 1b+2,resulted in breakthrough in protection at the highest challenge leveltested (9×10⁴) which was only slightly improved by adding domain 3.

[0087] The data indicates that the protective immunity induced by PA canbe attributed to individual domains (intact domain 1 and domain 4) or tocombinations of domains taken as permutations from all 4 domains.

[0088] The amino acid sequence and a DNA coding sequence for domain 4 isshown in FIG. 4 as SEQ ID NOs 15 and 16 respectively.

EXAMPLE 6

[0089] Further Testing of Domains as Vaccines

[0090] DNA encoding the PA domains, amino acids 1-259, 168-488, 1-488,168-596,1-596, 260-735, 489-735, 597-735 and 1-735 (truncates GST1,GST1b-2, GST1-2, GST1b-3, GST1-3, GST2-4, GST3-4, GST4 and GST1-4respectively) were PCR amplified from B. anthracis Sterne DNA and clonedin to the XhoI/BamHI sites of the expression vector pGEX-6-P3(Amersham-Pharmacia) downstream and in frame of the lac promoter.Proteins produced using this system were expressed as fusion proteinswith an N-terminal glutathione-s-transferase protein (GST). Recombinantplasmid DNA harbouring the DNA encoding the PA domains was thentransformed in to E. coli BL21 for protein expression studies.

[0091]E. Coli BL21 harbouring recombinant pGEX-6-P3 plasmids werecultured in L-broth containing 50 μg/ml ampicillin, 30 μg/mlchloramphenicol and 1% w/v glucose. Cultures were incubated with shaking(170 rev min⁻¹) at 30° C. to an A_(600 nm) 0.4, prior to induction with0.5 mM IPTG. Cultures were incubated for a further 4 hours, followed byharvesting by centrifugation at 10 000 rpm for 15 minutes.

[0092] Initial extraction of the PA truncates-fusion proteins indicatedthat they were produced as inclusion bodies. Cell pellets wereresuspended in phosphate buffered saline (PBS) and sonicated 4×20seconds in an iced water bath. The suspension was centrifuged at 15 000rpm for 15 minutes and cell pellets were then urea extracted, bysuspension in 8M urea with stirring at room temperature for 1 hour. Thesuspension was centrifuged for 15 minutes at 15000 rpm and thesupernatant dialysed against 100 mM Tris pH 8 containing 400 mML-arginine and 0.1 mM EDTA, prior to dialysis into PBS.

[0093] The successful refolding of the PA truncate-fusion proteinsallowed them to be purified on a glutathione Sepharose CL-4B affinitycolumn. All extracts (with the exception of truncate GST1b-2, amino acidresidues 168-487) were applied to a 15 ml glutathione Sepharose CL-4Bcolumn (Amersham-Parmacia), previously equilibrated with PBS andincubated, with rolling, overnight at 4° C. The column was washed withPBS and the fusion protein eluted with 50 mM Tris pH 7, containing 150mM NaCl, 1 mM EDTA and 20 mM reduced glutathione. Fractions containingthe PA truncates, identified by SDS-PAGE analysis, were pooled anddialysed against PBS. Protein concentration was determined using BCA(Perbio).

[0094] However truncate GST1b-2 could not be eluted from the glutathionesepharose CL-4B affinity column using reduced glutathione and wastherefore purified using ion exchange chromatography. Specifically,truncate GST1b-2 was dialysed against 20 mM Tris pH 8, prior to loadingonto a HiTrap Q column (Amersham-Parmacia), equilibrated with the samebuffer. Fusion protein was eluted with an increasing NaCl gradient of0-1M in 20 mM Tris pH8. Fractions containing the GST-protein werepooled, concentrated and loaded onto a HiLoad 26/60 Superdex 200 gelfiltration column (Amersham-Parmacia), previously equilibrated with PBS.Fractions containing fusion protein were pooled and the proteinconcentration determined by BCA (Perbio). Yields were between 1 and 43mg per litre of culture.

[0095] The molecular weight of the fragments and their recognition byantibodies to PA was confirmed using SDS PAGE and Western Blotting.Analysis of the rPA truncates by SDS Page and Western blotting showedprotein bands of the expected sizes. Some degradation in all of the rPAtruncates investigated was apparent showing similarity with recombinantPA expressed in B. subtilis. The rPA truncates GST1, GST1b-2 and GST1-2were particularly susceptible to degradation in the absence of domain 3.This has similarly been reported for rPA constructs containing mutationsin domain 3, that could not be purified from B. anthracis culturesupernatants (Brossier 1999), indicating that domain 3 may stabilisedomains 1 and 2.

[0096] Female, specific pathogen free A/J mice (Harlan UK) were used inthis study as these are a consistent model for anthrax infection (Welkos1986). Mice were age matched and seven weeks of age at the start of thestudy.

[0097] A/J mice were immunised on days 1 and 28 of the study with 10 μgof fusion protein adsorbed to 20% of 1.3% v/v Alhydrogel (HCI Biosector,Denmark) in a total volume of 100 μl of PBS. Groups immunised with rPAfrom B. subtilis (Miller 1998), with recombinant GST control protein, orfusion proteins encoding domains 1, 4 and 1-4 which had the GST tagremoved, were also included. Immunising doses were administeredintramuscularly into two sites on the hind legs. Mice were blood sampled37 days post primary immunisation for serum antibody analysis by enzymelinked immunosorbant assay (ELISA).

[0098] Microtitre plates (Immulon 2, Dynex Technologies) were coated,overnight at 4° C. with 5 μg/ml rPA, expressed from B. subtilis (Miller1998), in PBS except for two rows per plate which were coated with 5μg/ml anti-mouse Fab (Sigma, Poole, Dorset). Plates were washed with PBScontaining 1% v/v Tween 20 (PBS-T) and blocked with 5% w/v skimmed milkpowder in PBS (blotto) for 2 hours at 37° C. Serum, double-diluted in 1%blotto, was added to the rPA coated wells and was assayed in duplicatetogether with murine IgG standard (Sigma) added to the anti-fab coatedwells and incubated overnight at 4° C. After washing, horse-radishperoxidase conjugated goat anti-mouse IgG (Southern BiotechnologyAssociates Inc.), diluted 1 in 2000 in PBS, was added to all wells, andincubated for 1 hour at 37° C. Plates were washed again before additionof the substrate 2,2′-Azinobis (3-ethylbenzthiazoline-sulfonic acid)(1.09 mM ABTS, Sigma). After 20 minutes incubation at room temperature,the absorbance of the wells at 414 nm was measured (Titertek Multiscan,ICN Flow). Standard curves were calculated using Titersoft version 3.1csoftware. Titres were presented as μg IgG per ml serum and groupmeans±standard error of the mean (sem) were calculated. The results areshown in FIG. 5.

[0099] All the rPA truncates produced were immunogenic and stimulatedmean serum anti-rPA IgG concentrations in the A/J mice ranging from 6 μgper ml, for the GST1b-2 truncate immunised group, to 1488 μg per ml, inthe GST 1-4 truncate immunised group (FIG. 5). The GST control immunisedmice had no detectable antibodies to rPA.

[0100] Mice were challenged with B. anthracis STI spores on day 70 ofthe immunisation regimen. Sufficient STI spores for the challenge wereremoved from stock, washed in sterile distilled water and resuspended inPBS to a concentration of 1×10⁷ and 1×10⁶ spores per ml. Mice werechallenged intraperitoneally with 0.1 ml volumes containing 1×10⁶ and1×10⁵ spores per mouse, respectively, and were monitored for 14 day postchallenge to determine their protected status. Humane end-points werestrictly observed so that any animal displaying a collection of clinicalsigns which together indicated it had a lethal infection, was culled.The numbers of immunised mice which survived 14 days post challenge areshown in Table 3. TABLE 3 Challenge Level MLDs survivors/no. challenged(%) Domain 10² MLDs 10³ MLDs GST 1 3/5 (60) 1/5 (20)  GST 1b-2 1/5 (20)nd GST 1-2  5/5 (100) 3/5 (60)  GST 1b-3 3/5 (60) nd GST 1-3 4/5 (80) ndGST 1-4 nd 5/5 (100) GST 2-4 nd 5/5 (100) GST 3-4 nd 5/5 (100) GST 4 5/5 (100) 5/5 (100) GST 1 + GST 4 nd 5/5 (100) Cleaved 1 1/5 (20) 2/5Cleaved 4  5/5 (100) 5/5 Cleaved 1-4 nd 5/5 rPA nd 4/4 (100) control 0/5(0)  0/5 (0) 

[0101] The groups challenged with 10³ MLD's of STI spores were all fullyprotected except for the GST1, GST1-2 and cleaved 1 immunised groups inwhich there was some breakthrough in protection, and the control groupimmunised with GST only, which all succumbed to infection with a meantime to death (MTTD) of 2.4±0.2 days. At the lower challenge level of10² MLD's the GST1-2, GST4 and cleaved 4—immunised groups were all fullyprotected, but there was some breakthrough in protection in the othergroups. The mice that died in these groups had a MTTD of 4.5±0.2 dayswhich was not significantly different from the GST control immunisedgroup which all died with a MTTD of 4±0.4 days.

1 16 1 2228 DNA Artificial Sequence Description of Artificial SequenceNucleic acid which encodes the protective antigen of Bacillus anthracis1 aagcttcata tggaagtaaa gcaagagaac cgtctgctga acgaatctga atccagctct 60cagggcctgc ttggttacta tttctctgac ctgaacttcc aagcaccgat ggttgtaacc 120agctctacca ctggcgatct gtccatcccg tctagtgaac ttgagaacat tccaagcgag 180aaccagtatt tccagtctgc aatctggtcc ggttttatca aagtcaagaa atctgatgaa 240tacacgtttg ccacctctgc tgataaccac gtaaccatgt gggttgacga tcaggaagtg 300atcaacaaag catccaactc caacaaaatt cgtctggaaa aaggccgtct gtatcagatc 360aagattcagt accaacgcga gaacccgact gaaaaaggcc tggactttaa actgtattgg 420actgattctc agaacaagaa agaagtgatc agctctgaca atctgcaact gccggaattg 480aaacagaaaa gctccaactc tcgtaagaaa cgttccacca gcgctggccc gaccgtacca 540gatcgcgaca acgatggtat tccggactct ctggaagttg aaggctacac ggttgatgta 600aagaacaaac gtaccttcct tagtccgtgg atctccaata ttcacgagaa gaaaggtctg 660accaaataca aatccagtcc ggaaaaatgg tccactgcat ctgatccgta ctctgacttt 720gagaaagtga ccggtcgtat cgacaagaac gtctctccgg aagcacgcca tccactggtt 780gctgcgtatc cgatcgtaca tgttgacatg gaaaacatca ttttgtccaa gaacgaagac 840cagtccactc agaacactga ctctgaaact cgtaccatct ccaagaacac ctccacgtct 900cgtactcaca ccagtgaagt acatggtaac gctgaagtac acgcctcttt ctttgacatc 960ggcggctctg ttagcgctgg cttctccaac tctaattctt ctactgttgc cattgatcac 1020tctctgagtc tggctggcga acgtacctgg gcagagacca tgggtcttaa cactgctgat 1080accgcgcgtc tgaatgctaa cattcgctac gtcaacactg gtacggcacc gatctacaac 1140gtactgccaa ccaccagcct ggttctgggt aagaaccaga ctcttgcgac catcaaagcc 1200aaagagaacc aactgtctca gattctggca ccgaataact actatccttc caagaacctg 1260gctccgatcg cactgaacgc acaggatgac ttctcttcca ctccgatcac catgaactac 1320aaccagttcc tggaacttga gaagaccaaa cagctgcgtc ttgacactga ccaagtgtac 1380ggtaacatcg cgacctacaa ctttgagaac ggtcgcgtcc gcgttgacac aggctctaat 1440tggtctgaag tactgcctca gattcaggaa accaccgctc gtatcatctt caacggtaaa 1500gacctgaacc tggttgaacg tcgtattgct gctgtgaacc cgtctgatcc attagagacc 1560accaaaccgg atatgactct gaaagaagcc ctgaagatcg cctttggctt caacgagccg 1620aacggtaatc ttcagtacca aggtaaagac atcactgaat ttgacttcaa ctttgatcag 1680cagacctctc agaatatcaa gaaccaactg gctgagctga acgcgaccaa tatctatacg 1740gtactcgaca agatcaaact gaacgcgaaa atgaacattc tgattcgcga caaacgtttc 1800cactacgatc gtaataacat cgctgttggc gctgatgaat ctgttgtgaa agaagcgcat 1860cgcgaagtca tcaactccag caccgaaggc ctgcttctga acatcgacaa agacattcgt 1920aagatcctgt ctggttacat tgttgagatc gaagacaccg aaggcctgaa agaagtgatc 1980aatgatcgtt acgacatgct gaacatcagc tctctgcgtc aagatggtaa gacgttcatt 2040gacttcaaga aatacaacga caaacttccg ctgtatatct ctaatccgaa ctacaaagtg 2100aacgtttacg ctgttaccaa agagaacacc atcatcaatc catctgagaa cggcgatacc 2160tctaccaacg gtatcaagaa gattctgatc ttctccaaga aaggttacga gatcggttaa 2220taggatcc 2228 2 10 PRT Bacillus anthracis 2 Met Glu Val Lys Gln Glu AsnArg Leu Leu 1 5 10 3 258 PRT Bacillus anthracis 3 Glu Val Lys Gln GluAsn Arg Leu Leu Asn Glu Ser Glu Ser Ser Ser 1 5 10 15 Gln Gly Leu LeuGly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30 Met Val Val ThrSer Ser Thr Thr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45 Glu Leu Glu AsnIle Pro Ser Glu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60 Trp Ser Gly PheIle Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala 65 70 75 80 Thr Ser AlaAsp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95 Ile Asn LysAla Ser Asn Ser Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110 Leu TyrGln Ile Lys Ile Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125 GlyLeu Asp Phe Lys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser 145 150155 160 Ser Asn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro165 170 175 Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu GlyTyr 180 185 190 Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro TrpIle Ser 195 200 205 Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys SerSer Pro Glu 210 215 220 Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp PheGlu Lys Val Thr 225 230 235 240 Gly Arg Ile Asp Lys Asn Val Ser Pro GluAla Arg His Pro Leu Val 245 250 255 Ala Ala 4 774 DNA ArtificialSequence Description of Artificial Sequence DNA sequence used to encodeSEQ ID NO 3 4 gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttcccaggggttacta 60 ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacctcttctactaca 120 ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaaccaatatttt 180 caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaatatacatttgct 240 acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgattaataaagct 300 tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaaaattcaatat 360 caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggaccgattctcaa 420 aataaaaaag aagtgatttc tagtgataac ttacaattgc cagaattaaaacaaaaatct 480 tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccagaccgtgacaat 540 gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaaaaataaaaga 600 acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaaccaaatataaa 660 tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcgaaaaggttaca 720 ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggcagct 774 5 487 PRT Artificial Sequence Description of ArtificialSequence Fusion protein 5 Glu Val Lys Gln Glu Asn Arg Leu Leu Asn GluSer Glu Ser Ser Ser 1 5 10 15 Gln Gly Leu Leu Gly Tyr Tyr Phe Ser AspLeu Asn Phe Gln Ala Pro 20 25 30 Met Val Val Thr Ser Ser Thr Thr Gly AspLeu Ser Ile Pro Ser Ser 35 40 45 Glu Leu Glu Asn Ile Pro Ser Glu Asn GlnTyr Phe Gln Ser Ala Ile 50 55 60 Trp Ser Gly Phe Ile Lys Val Lys Lys SerAsp Glu Tyr Thr Phe Ala 65 70 75 80 Thr Ser Ala Asp Asn His Val Thr MetTrp Val Asp Asp Gln Glu Val 85 90 95 Ile Asn Lys Ala Ser Asn Ser Asn LysIle Arg Leu Glu Lys Gly Arg 100 105 110 Leu Tyr Gln Ile Lys Ile Gln TyrGln Arg Glu Asn Pro Thr Glu Lys 115 120 125 Gly Leu Asp Phe Lys Leu TyrTrp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140 Val Ile Ser Ser Asp AsnLeu Gln Leu Pro Glu Leu Lys Gln Lys Ser 145 150 155 160 Ser Asn Ser ArgLys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175 Asp Arg AspAsn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185 190 Thr ValAsp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195 200 205 AsnIle His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu 210 215 220Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys Val Thr 225 230235 240 Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His Pro Leu Val245 250 255 Ala Ala Tyr Pro Ile Val His Val Asp Met Glu Asn Ile Ile LeuSer 260 265 270 Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr Asp Ser Glu ThrArg Thr 275 280 285 Ile Ser Lys Asn Thr Ser Thr Ser Arg Thr His Thr SerGlu Val His 290 295 300 Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp IleGly Gly Ser Val 305 310 315 320 Ser Ala Gly Phe Ser Asn Ser Asn Ser SerThr Val Ala Ile Asp His 325 330 335 Ser Leu Ser Leu Ala Gly Glu Arg ThrTrp Ala Glu Thr Met Gly Leu 340 345 350 Asn Thr Ala Asp Thr Ala Arg LeuAsn Ala Asn Ile Arg Tyr Val Asn 355 360 365 Thr Gly Thr Ala Pro Ile TyrAsn Val Leu Pro Thr Thr Ser Leu Val 370 375 380 Leu Gly Lys Asn Gln ThrLeu Ala Thr Ile Lys Ala Lys Glu Asn Gln 385 390 395 400 Leu Ser Gln IleLeu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415 Ala Pro IleAla Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425 430 Thr MetAsn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435 440 445 ArgLeu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe 450 455 460Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser Glu Val 465 470475 480 Leu Pro Gln Ile Gln Glu Thr 485 6 1461 DNA Artificial SequenceDescription of Artificial Sequence DNA sequence used to encode SEQ ID NO5 6 gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacttc ttctactaca 120ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420aataaaaaag aagtgatttc tagtgataac ttacaactgc cagaattaaa acaaaaatct 480tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agcttatccg 780attgtacatg tagatatgga gaatattatt ctctcaaaaa atgaggatca atccacacag 840aatactgata gtgaaacgag aacaataagt aaaaatactt ctacaagtag gacacatact 900agtgaagtac atggaaatgc agaagtgcat gcgtcgttct ttgatattgg tgggagtgta 960tctgcaggat ttagtaattc gaattcaagt acggtcgcaa ttgatcattc actatctcta 1020gcaggggaaa gaacttgggc tgaaacaatg ggtttaaata ccgctgatac agcaagatta 1080aatgccaata ttagatatgt aaatactggg acggctccaa tctacaacgt gttaccaacg 1140acttcgttag tgttaggaaa aaatcaaaca ctcgcgacaa ttaaagctaa ggaaaaccaa 1200ttaagtcaaa tacttgcacc taataattat tatccttcta aaaacttggc gccaatcgca 1260ttaaatgcac aagacgattt cagttctact ccaattacaa tgaattacaa tcaatttctt 1320gagttagaaa aaacgaaaca attaagatta gatacggatc aagtatatgg gaatatagca 1380acatacaatt ttgaaaatgg aagagtgagg gtggatacag gctcgaactg gagtgaagtg 1440ttaccgcaaa ttcaagaaac a 1461 7 318 PRT Artificial Sequence Descriptionof Artificial Sequence Fusion protein 7 Ser Ala Gly Pro Thr Val Pro AspArg Asp Asn Asp Gly Ile Pro Asp 1 5 10 15 Ser Leu Glu Val Glu Gly TyrThr Val Asp Val Lys Asn Lys Arg Thr 20 25 30 Phe Leu Ser Pro Trp Ile SerAsn Ile His Glu Lys Lys Gly Leu Thr 35 40 45 Lys Tyr Lys Ser Ser Pro GluLys Trp Ser Thr Ala Ser Asp Pro Tyr 50 55 60 Ser Asp Phe Glu Lys Val ThrGly Arg Ile Asp Lys Asn Val Ser Pro 65 70 75 80 Glu Ala Arg His Pro LeuVal Ala Ala Tyr Pro Ile Val His Val Asp 85 90 95 Met Glu Asn Ile Ile LeuSer Lys Asn Glu Asp Gln Ser Thr Gln Asn 100 105 110 Thr Asp Ser Glu ThrArg Thr Ile Ser Lys Asn Thr Ser Thr Ser Arg 115 120 125 Thr His Thr SerGlu Val His Gly Asn Ala Glu Val His Ala Ser Phe 130 135 140 Phe Asp IleGly Gly Ser Val Ser Ala Gly Phe Ser Asn Ser Asn Ser 145 150 155 160 SerThr Val Ala Ile Asp His Ser Leu Ser Leu Ala Gly Glu Arg Thr 165 170 175Trp Ala Glu Thr Met Gly Leu Asn Thr Ala Asp Thr Ala Arg Leu Asn 180 185190 Ala Asn Ile Arg Tyr Val Asn Thr Gly Thr Ala Pro Ile Tyr Asn Val 195200 205 Leu Pro Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr210 215 220 Ile Lys Ala Lys Glu Asn Gln Leu Ser Gln Ile Leu Ala Pro AsnAsn 225 230 235 240 Tyr Tyr Pro Ser Lys Asn Leu Ala Pro Ile Ala Leu AsnAla Gln Asp 245 250 255 Asp Phe Ser Ser Thr Pro Ile Thr Met Asn Tyr AsnGln Phe Leu Glu 260 265 270 Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp ThrAsp Gln Val Tyr Gly 275 280 285 Asn Ile Ala Thr Tyr Asn Phe Glu Asn GlyArg Val Arg Val Asp Thr 290 295 300 Gly Ser Asn Trp Ser Glu Val Leu ProGln Ile Gln Glu Thr 305 310 315 8 954 DNA Artificial SequenceDescription of Artificial Sequence DNA sequence used to encode SEQ ID NO7 8 agtgctggac ctacggttcc agaccgtgac aatgatggaa tccctgattc attagaggta 60gaaggatata cggttgatgt caaaaataaa agaacttttc tttcaccatg gatttctaat 120attcatgaaa agaaaggatt aaccaaatat aaatcatctc ctgaaaaatg gagcacggct 180tctgatccgt acagtgattt cgaaaaggtt acaggacgga ttgataagaa tgtatcacca 240gaggcaagac acccccttgt ggcagcttat ccgattgtac atgtagatat ggagaatatt 300attctctcaa aaaatgagga tcaatccaca cagaatactg atagtgaaac gagaacaata 360agtaaaaata cttctacaag taggacacat actagtgaag tacatggaaa tgcagaagtg 420catgcgtcgt tctttgatat tggtgggagt gtatctgcag gatttagtaa ttcgaattca 480agtacggtcg caattgatca ttcactatct ctagcagggg aaagaacttg ggctgaaaca 540atgggtttaa ataccgctga tacagcaaga ttaaatgcca atattagata tgtaaatact 600gggacggctc caatctacaa cgtgttacca acgacttcgt tagtgttagg aaaaaatcaa 660acactcgcga caattaaagc taaggaaaac caattaagtc aaatacttgc acctaataat 720tattatcctt ctaaaaactt ggcgccaatc gcattaaatg cacaagacga tttcagttct 780actccaatta caatgaatta caatcaattt cttgagttag aaaaaacgaa acaattaaga 840ttagatacgg atcaagtata tgggaatata gcaacataca attttgaaaa tggaagagtg 900agggtggata caggctcgaa ctggagtgaa gtgttaccgc aaattcaaga aaca 954 9 426PRT Artificial Sequence Description of Artificial Sequence Fusionprotein 9 Ser Ala Gly Pro Thr Val Pro Asp Arg Asp Asn Asp Gly Ile ProAsp 1 5 10 15 Ser Leu Glu Val Glu Gly Tyr Thr Val Asp Val Lys Asn LysArg Thr 20 25 30 Phe Leu Ser Pro Trp Ile Ser Asn Ile His Glu Lys Lys GlyLeu Thr 35 40 45 Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser AspPro Tyr 50 55 60 Ser Asp Phe Glu Lys Val Thr Gly Arg Ile Asp Lys Asn ValSer Pro 65 70 75 80 Glu Ala Arg His Pro Leu Val Ala Ala Tyr Pro Ile ValHis Val Asp 85 90 95 Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp Gln SerThr Gln Asn 100 105 110 Thr Asp Ser Glu Thr Arg Thr Ile Ser Lys Asn ThrSer Thr Ser Arg 115 120 125 Thr His Thr Ser Glu Val His Gly Asn Ala GluVal His Ala Ser Phe 130 135 140 Phe Asp Ile Gly Gly Ser Val Ser Ala GlyPhe Ser Asn Ser Asn Ser 145 150 155 160 Ser Thr Val Ala Ile Asp His SerLeu Ser Leu Ala Gly Glu Arg Thr 165 170 175 Trp Ala Glu Thr Met Gly LeuAsn Thr Ala Asp Thr Ala Arg Leu Asn 180 185 190 Ala Asn Ile Arg Tyr ValAsn Thr Gly Thr Ala Pro Ile Tyr Asn Val 195 200 205 Leu Pro Thr Thr SerLeu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr 210 215 220 Ile Lys Ala LysGlu Asn Gln Leu Ser Gln Ile Leu Ala Pro Asn Asn 225 230 235 240 Tyr TyrPro Ser Lys Asn Leu Ala Pro Ile Ala Leu Asn Ala Gln Asp 245 250 255 AspPhe Ser Ser Thr Pro Ile Thr Met Asn Tyr Asn Gln Phe Leu Glu 260 265 270Leu Glu Lys Thr Lys Gln Leu Arg Leu Asp Thr Asp Gln Val Tyr Gly 275 280285 Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly Arg Val Arg Val Asp Thr 290295 300 Gly Ser Asn Trp Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr Ala305 310 315 320 Arg Ile Ile Phe Asn Gly Lys Asp Leu Asn Leu Val Glu ArgArg Ile 325 330 335 Ala Ala Val Asn Pro Ser Asp Pro Leu Glu Thr Thr LysPro Asp Met 340 345 350 Thr Leu Lys Glu Ala Leu Lys Ile Ala Phe Gly PheAsn Glu Pro Asn 355 360 365 Gly Asn Leu Gln Tyr Gln Gly Lys Asp Ile ThrGlu Phe Asp Phe Asn 370 375 380 Phe Asp Gln Gln Thr Ser Gln Asn Ile LysAsn Gln Leu Ala Glu Leu 385 390 395 400 Asn Ala Thr Asn Ile Tyr Thr ValLeu Asp Lys Ile Lys Leu Asn Ala 405 410 415 Lys Met Asn Ile Leu Ile ArgAsp Lys Arg 420 425 10 1278 DNA Artificial Sequence Description ofArtificial Sequence DNA sequence used to encode SEQ ID NO 9 10agtgctggac ctacggttcc agaccgtgac aatgatggaa tccctgattc attagaggta 60gaaggatata cggttgatgt caaaaataaa agaacttttc tttcaccatg gatttctaat 120attcatgaaa agaaaggatt aaccaaatat aaatcatctc ctgaaaaatg gagcacggct 180tctgatccgt acagtgattt cgaaaaggtt acaggacgga ttgataagaa tgtatcacca 240gaggcaagac acccccttgt ggcagcttat ccgattgtac atgtagatat ggagaatatt 300attctctcaa aaaatgagga tcaatccaca cagaatactg atagtgaaac gagaacaata 360agtaaaaata cttctacaag taggacacat actagtgaag tacatggaaa tgcagaagtg 420catgcgtcgt tctttgatat tggtgggagt gtatctgcag gatttagtaa ttcgaattca 480agtacggtcg caattgatca ttcactatct ctagcagggg aaagaacttg ggctgaaaca 540atgggtttaa ataccgctga tacagcaaga ttaaatgcca atattagata tgtaaatact 600gggacggctc caatctacaa cgtgttacca acgacttcgt tagtgttagg aaaaaatcaa 660acactcgcga caattaaagc taaggaaaac caattaagtc aaatacttgc acctaataat 720tattatcctt ctaaaaactt ggcgccaatc gcattaaatg cacaagacga tttcagttct 780actccaatta caatgaatta caatcaattt cttgagttag aaaaaacgaa acaattaaga 840ttagatacgg atcaagtata tgggaatata gcaacataca attttgaaaa tggaagagtg 900agggtggata caggctcgaa ctggagtgaa gtgttaccgc aaattcaaga aacaactgca 960cgtatcattt ttaatggaaa agatttaaat ctggtagaaa ggcggatagc ggcggttaat 1020cctagtgatc cattagaaac gactaaaccg gatatgacat taaaagaagc ccttaaaata 1080gcatttggat ttaacgaacc gaatggaaac ttacaatatc aagggaaaga cataaccgaa 1140tttgatttta atttcgatca acaaacatct caaaatatca agaatcagtt agcggaatta 1200aacgcaacta acatatatac tgtattagat aaaatcaaat taaatgcaaa aatgaatatt 1260ttaataagag ataaacgt 1278 11 595 PRT Artificial Sequence Description ofArtificial Sequence Fusion protein 11 Glu Val Lys Gln Glu Asn Arg LeuLeu Asn Glu Ser Glu Ser Ser Ser 1 5 10 15 Gln Gly Leu Leu Gly Tyr TyrPhe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30 Met Val Val Thr Ser Ser ThrThr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45 Glu Leu Glu Asn Ile Pro SerGlu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60 Trp Ser Gly Phe Ile Lys ValLys Lys Ser Asp Glu Tyr Thr Phe Ala 65 70 75 80 Thr Ser Ala Asp Asn HisVal Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95 Ile Asn Lys Ala Ser AsnSer Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110 Leu Tyr Gln Ile LysIle Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125 Gly Leu Asp PheLys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140 Val Ile SerSer Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser 145 150 155 160 SerAsn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185190 Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195200 205 Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220 Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys ValThr 225 230 235 240 Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg HisPro Leu Val 245 250 255 Ala Ala Tyr Pro Ile Val His Val Asp Met Glu AsnIle Ile Leu Ser 260 265 270 Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr AspSer Glu Thr Arg Thr 275 280 285 Ile Ser Lys Asn Thr Ser Thr Ser Arg ThrHis Thr Ser Glu Val His 290 295 300 Gly Asn Ala Glu Val His Ala Ser PhePhe Asp Ile Gly Gly Ser Val 305 310 315 320 Ser Ala Gly Phe Ser Asn SerAsn Ser Ser Thr Val Ala Ile Asp His 325 330 335 Ser Leu Ser Leu Ala GlyGlu Arg Thr Trp Ala Glu Thr Met Gly Leu 340 345 350 Asn Thr Ala Asp ThrAla Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn 355 360 365 Thr Gly Thr AlaPro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val 370 375 380 Leu Gly LysAsn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln 385 390 395 400 LeuSer Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425430 Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435440 445 Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460 Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser GluVal 465 470 475 480 Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile PheAsn Gly Lys 485 490 495 Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala ValAsn Pro Ser Asp 500 505 510 Pro Leu Glu Thr Thr Lys Pro Asp Met Thr LeuLys Glu Ala Leu Lys 515 520 525 Ile Ala Phe Gly Phe Asn Glu Pro Asn GlyAsn Leu Gln Tyr Gln Gly 530 535 540 Lys Asp Ile Thr Glu Phe Asp Phe AsnPhe Asp Gln Gln Thr Ser Gln 545 550 555 560 Asn Ile Lys Asn Gln Leu AlaGlu Leu Asn Ala Thr Asn Ile Tyr Thr 565 570 575 Val Leu Asp Lys Ile LysLeu Asn Ala Lys Met Asn Ile Leu Ile Arg 580 585 590 Asp Lys Arg 595 121785 DNA Artificial Sequence Description of Artificial Sequence DNAsequence used to encode SEQ ID NO 11 12 gaagttaaac aggagaaccg gttattaaatgaatcagaat caagttccca ggggttacta 60 ggatactatt ttagtgattt gaattttcaagcacccatgg tggttacctc ttctactaca 120 ggggatttat ctattcctag ttctgagttagaaaatattc catcggaaaa ccaatatttt 180 caatctgcta tttggtcagg atttatcaaagttaagaaga gtgatgaata tacatttgct 240 acttccgctg ataatcatgt aacaatgtgggtagatgacc aagaagtgat taataaagct 300 tctaattcta acaaaatcag attagaaaaaggaagattat atcaaataaa aattcaatat 360 caacgagaaa atcctactga aaaaggattggatttcaagt tgtactggac cgattctcaa 420 aataaaaaag aagtgatttc tagtgataacttacaattgc cagaattaaa acaaaaatct 480 tcgaactcaa gaaaaaagcg aagtacaagtgctggaccta cggttccaga ccgtgacaat 540 gatggaatcc ctgattcatt agaggtagaaggatatacgg ttgatgtcaa aaataaaaga 600 acttttcttt caccatggat ttctaatattcatgaaaaga aaggattaac caaatataaa 660 tcatctcctg aaaaatggag cacggcttctgatccgtaca gtgatttcga aaaggttaca 720 ggacggattg ataagaatgt atcaccagaggcaagacacc cccttgtggc agcttatccg 780 attgtacatg tagatatgga gaatattattctctcaaaaa atgaggatca atccacacag 840 aatactgata gtgaaacgag aacaataagtaaaaatactt ctacaagtag gacacatact 900 agtgaagtac atggaaatgc agaagtgcatgcgtcgttct ttgatattgg tgggagtgta 960 tctgcaggat ttagtaattc gaattcaagtacggtcgcaa ttgatcattc actatctcta 1020 gcaggggaaa gaacttgggc tgaaacaatgggtttaaata ccgctgatac agcaagatta 1080 aatgccaata ttagatatgt aaatactgggacggctccaa tctacaacgt gttaccaacg 1140 acttcgttag tgttaggaaa aaatcaaacactcgcgacaa ttaaagctaa ggaaaaccaa 1200 ttaagtcaaa tacttgcacc taataattattatccttcta aaaacttggc gccaatcgca 1260 ttaaatgcac aagacgattt cagttctactccaattacaa tgaattacaa tcaatttctt 1320 gagttagaaa aaacgaaaca attaagattagatacggatc aagtatatgg gaatatagca 1380 acatacaatt ttgaaaatgg aagagtgagggtggatacag gctcgaactg gagtgaagtg 1440 ttaccgcaaa ttcaagaaac aactgcacgtatcattttta atggaaaaga tttaaatctg 1500 gtagaaaggc ggatagcggc ggttaatcctagtgatccat tagaaacgac taaaccggat 1560 atgacattaa aagaagccct taaaatagcatttggattta acgaaccgaa tggaaactta 1620 caatatcaag ggaaagacat aaccgaatttgattttaatt tcgatcaaca aacatctcaa 1680 aatatcaaga atcagttagc ggaattaaacgcaactaaca tatatactgt attagataaa 1740 atcaaattaa atgcaaaaat gaatattttaataagagata aacgt 1785 13 735 PRT Artificial Sequence Description ofArtificial Sequence Fusion protein 13 Glu Val Lys Gln Glu Asn Arg LeuLeu Asn Glu Ser Glu Ser Ser Ser 1 5 10 15 Gln Gly Leu Leu Gly Tyr TyrPhe Ser Asp Leu Asn Phe Gln Ala Pro 20 25 30 Met Val Val Thr Ser Ser ThrThr Gly Asp Leu Ser Ile Pro Ser Ser 35 40 45 Glu Leu Glu Asn Ile Pro SerGlu Asn Gln Tyr Phe Gln Ser Ala Ile 50 55 60 Trp Ser Gly Phe Ile Lys ValLys Lys Ser Asp Glu Tyr Thr Phe Ala 65 70 75 80 Thr Ser Ala Asp Asn HisVal Thr Met Trp Val Asp Asp Gln Glu Val 85 90 95 Ile Asn Lys Ala Ser AsnSer Asn Lys Ile Arg Leu Glu Lys Gly Arg 100 105 110 Leu Tyr Gln Ile LysIle Gln Tyr Gln Arg Glu Asn Pro Thr Glu Lys 115 120 125 Gly Leu Asp PheLys Leu Tyr Trp Thr Asp Ser Gln Asn Lys Lys Glu 130 135 140 Val Ile SerSer Asp Asn Leu Gln Leu Pro Glu Leu Lys Gln Lys Ser 145 150 155 160 SerAsn Ser Arg Lys Lys Arg Ser Thr Ser Ala Gly Pro Thr Val Pro 165 170 175Asp Arg Asp Asn Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr 180 185190 Thr Val Asp Val Lys Asn Lys Arg Thr Phe Leu Ser Pro Trp Ile Ser 195200 205 Asn Ile His Glu Lys Lys Gly Leu Thr Lys Tyr Lys Ser Ser Pro Glu210 215 220 Lys Trp Ser Thr Ala Ser Asp Pro Tyr Ser Asp Phe Glu Lys ValThr 225 230 235 240 Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg HisPro Leu Val 245 250 255 Ala Ala Tyr Pro Ile Val His Val Asp Met Glu AsnIle Ile Leu Ser 260 265 270 Lys Asn Glu Asp Gln Ser Thr Gln Asn Thr AspSer Gln Thr Arg Thr 275 280 285 Ile Ser Lys Asn Thr Ser Thr Ser Arg ThrHis Thr Ser Glu Val His 290 295 300 Gly Asn Ala Glu Val His Ala Ser PhePhe Asp Ile Gly Gly Ser Val 305 310 315 320 Ser Ala Gly Phe Ser Asn SerAsn Ser Ser Thr Val Ala Ile Asp His 325 330 335 Ser Leu Ser Leu Ala GlyGlu Arg Thr Trp Ala Glu Thr Met Gly Leu 340 345 350 Asn Thr Ala Asp ThrAla Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn 355 360 365 Thr Gly Thr AlaPro Ile Tyr Asn Val Leu Pro Thr Thr Ser Leu Val 370 375 380 Leu Gly LysAsn Gln Thr Leu Ala Thr Ile Lys Ala Lys Glu Asn Gln 385 390 395 400 LeuSer Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu 405 410 415Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe Ser Ser Thr Pro Ile 420 425430 Thr Met Asn Tyr Asn Gln Phe Leu Glu Leu Glu Lys Thr Lys Gln Leu 435440 445 Arg Leu Asp Thr Asp Gln Val Tyr Gly Asn Ile Ala Thr Tyr Asn Phe450 455 460 Glu Asn Gly Arg Val Arg Val Asp Thr Gly Ser Asn Trp Ser GluVal 465 470 475 480 Leu Pro Gln Ile Gln Glu Thr Thr Ala Arg Ile Ile PheAsn Gly Lys 485 490 495 Asp Leu Asn Leu Val Glu Arg Arg Ile Ala Ala ValAsn Pro Ser Asp 500 505 510 Pro Leu Glu Thr Thr Lys Pro Asp Met Thr LeuLys Glu Ala Leu Lys 515 520 525 Ile Ala Phe Gly Phe Asn Glu Pro Asn GlyAsn Leu Gln Tyr Gln Gly 530 535 540 Lys Asp Ile Thr Glu Phe Asp Phe AsnPhe Asp Gln Gln Thr Ser Gln 545 550 555 560 Asn Ile Lys Asn Gln Leu AlaGlu Leu Asn Ala Thr Asn Ile Tyr Thr 565 570 575 Val Leu Asp Lys Ile LysLeu Asn Ala Lys Met Asn Ile Leu Ile Arg 580 585 590 Asp Lys Arg Phe HisTyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp 595 600 605 Glu Ser Val ValLys Glu Ala His Arg Glu Val Ile Asn Ser Ser Thr 610 615 620 Glu Gly LeuLeu Leu Asn Ile Asp Lys Asp Ile Arg Lys Ile Leu Ser 625 630 635 640 GlyTyr Ile Val Glu Ile Glu Asp Thr Glu Gly Leu Lys Glu Val Ile 645 650 655Asn Asp Arg Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg Gln Asp Gly 660 665670 Lys Thr Phe Ile Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr 675680 685 Ile Ser Asn Pro Asn Tyr Lys Val Asn Val Tyr Ala Val Thr Lys Glu690 695 700 Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr Ser Thr AsnGly 705 710 715 720 Ile Lys Lys Ile Leu Ile Phe Ser Lys Lys Gly Tyr GluIle Gly 725 730 735 14 2208 DNA Artificial Sequence Description ofArtificial Sequence DNA sequence used to encode SEQ ID NO 13 14gaagttaaac aggagaaccg gttattaaat gaatcagaat caagttccca ggggttacta 60ggatactatt ttagtgattt gaattttcaa gcacccatgg tggttacctc ttctactaca 120ggggatttat ctattcctag ttctgagtta gaaaatattc catcggaaaa ccaatatttt 180caatctgcta tttggtcagg atttatcaaa gttaagaaga gtgatgaata tacatttgct 240acttccgctg ataatcatgt aacaatgtgg gtagatgacc aagaagtgat taataaagct 300tctaattcta acaaaatcag attagaaaaa ggaagattat atcaaataaa aattcaatat 360caacgagaaa atcctactga aaaaggattg gatttcaagt tgtactggac cgattctcaa 420aataaaaaag aagtgatttc tagtgataac ttacaattgc cagaattaaa acaaaaatct 480tcgaactcaa gaaaaaagcg aagtacaagt gctggaccta cggttccaga ccgtgacaat 540gatggaatcc ctgattcatt agaggtagaa ggatatacgg ttgatgtcaa aaataaaaga 600acttttcttt caccatggat ttctaatatt catgaaaaga aaggattaac caaatataaa 660tcatctcctg aaaaatggag cacggcttct gatccgtaca gtgatttcga aaaggttaca 720ggacggattg ataagaatgt atcaccagag gcaagacacc cccttgtggc agcttatccg 780attgtacatg tagatatgga gaatattatt ctctcaaaaa atgaggatca atccacacag 840aatactgata gtgaaacgag aacaataagt aaaaatactt ctacaagtag gacacatact 900agtgaagtac atggaaatgc agaagtgcat gcgtcgttct ttgatattgg tgggagtgta 960tctgcaggat ttagtaattc gaattcaagt acggtcgcaa ttgatcattc actatctcta 1020gcaggggaaa gaacttgggc tgaaacaatg ggtttaaata ccgctgatac agcaagatta 1080aatgccaata ttagatatgt aaatactggg acggctccaa tctacaacgt gttaccaacg 1140acttcgttag tgttaggaaa aaatcaaaca ctcgcgacaa ttaaagctaa ggaaaaccaa 1200ttaagtcaaa tacttgcacc taataattat tatccttcta aaaacttggc gccaatcgca 1260ttaaatgcac aagacgattt cagttctact ccaattacaa tgaattacaa tcaatttctt 1320gagttagaaa aaacgaaaca attaagatta gatacggatc aagtatatgg gaatatagca 1380acatacaatt ttgaaaatgg aagagtgagg gtggatacag gctcgaactg gagtgaagtg 1440ttaccgcaaa ttcaagaaac aactgcacgt atcattttta atggaaaaga tttaaatctg 1500gtagaaaggc ggatagcggc ggttaatcct agtgatccat tagaaacgac taaaccggat 1560atgacattaa aagaagccct taaaatagca tttggattta acgaaccgaa tggaaactta 1620caatatcaag ggaaagacat aaccgaattt gattttaatt tcgatcaaca aacatctcaa 1680aatatcaaga atcagttagc ggaattaaac gcaactaaca tatatactgt attagataaa 1740atcaaattaa atgcaaaaat gaatatttta ataagagata aacgttttca ttatgataga 1800aataacatag cagttggggc ggatgagtca gtagttaagg aggctcatag agaagtaatt 1860aattcgtcaa cagagggatt attgttaaat attgataagg atataagaaa aatattatca 1920ggttatattg tagaaattga agatactgaa gggcttaaag aagttataaa tgacagatat 1980gatatgttga atatttctag tttacggcaa gatggaaaaa catttataga ttttaaaaaa 2040tataatgata aattaccgtt atatataagt aatcccaatt ataaggtaaa tgtatatgct 2100gttactaaag aaaacactat tattaatcct agtgagaatg gggatactag taccaacggg 2160atcaagaaaa ttttaatctt ttctaaaaaa ggctatgaga taggataa 2208 15 140 PRTBacillus anthracis 15 Phe His Tyr Asp Arg Asn Asn Ile Ala Val Gly AlaAsp Glu Ser Val 1 5 10 15 Val Lys Glu Ala His Arg Glu Val Ile Asn SerSer Thr Glu Gly Leu 20 25 30 Leu Leu Asn Ile Asp Lys Asp Ile Arg Lys IleLeu Ser Gly Tyr Ile 35 40 45 Val Glu Ile Glu Asp Thr Glu Gly Leu Lys GluVal Ile Asn Asp Arg 50 55 60 Tyr Asp Met Leu Asn Ile Ser Ser Leu Arg GlnAsp Gly Lys Thr Phe 65 70 75 80 Ile Asp Phe Lys Lys Tyr Asn Asp Lys LeuPro Leu Tyr Ile Ser Asn 85 90 95 Pro Asn Tyr Lys Val Asn Val Tyr Ala ValThr Lys Glu Asn Thr Ile 100 105 110 Ile Asn Pro Ser Glu Asn Gly Asp ThrSer Thr Asn Gly Ile Lys Lys 115 120 125 Ile Leu Ile Phe Ser Lys Lys GlyTyr Glu Ile Gly 130 135 140 16 423 DNA Artificial Sequence Descriptionof Artificial Sequence DNA coding sequence for domain 4. 16 tttcattatgatagaaataa catagcagtt ggggcggatg agtcagtagt taaggaggct 60 catagagaagtaattaattc gtcaacagag ggattattgt taaatattga taaggatata 120 agaaaaatattatcaggtta tattgtagaa attgaagata ctgaagggct taaagaagtt 180 ataaatgacagatatgatat gttgaatatt tctagtttac ggcaagatgg aaaaacattt 240 atagattttaaaaaatataa tgataaatta ccgttatata taagtaatcc caattataag 300 gtaaatgtatatgctgttac taaagaaaac actattatta atcctagtga gaatggggat 360 actagtaccaacgggatcaa gaaaatttta atcttttcta aaaaaggcta tgagatagga 420 taa 423

1. An immunogenic reagent which produces an immune response which isprotective against Bacillus anthracis, said reagent comprising one ormore polypeptides which together represent up to three domains of thefull length Protective Antigen (PA) of B. anthracis or variants ofthese, and at least one of said domains comprises domain 1 or domain 4of PA or a variant thereof.
 2. An immunogenic reagent according to claim1 which comprises domain 4 of the PA of B. anthracis.
 3. An immunogenicreagent according to claim 1 or claim 2 which comprises a combination ofdomains 1 and 4 or protective regions thereof.
 4. An immunogenic reagentaccording to claim 3 which comprises the sequence of domain 1 and domain4 of wild-type PA.
 5. An immunogenic reagent according to claim 4wherein said domains are present in the form of a fusion polypeptide. 6.An immunogenic reagent according to claim 5 which comprises domain 1fused to domain 2 of the PA sequence.
 7. An immunogenic reagentaccording to claim 6 which is fused to domain 3 of the PA sequence. 8.An immunogenic reagent according to claim 4 which comprises a mixture ofa polypeptides, one of which comprises domain 1 and one of whichcomprises domain 4 of the PA sequence.
 9. An immunogenic reagentaccording to any one of the preceding claims wherein a polypeptide isfused to a further polypeptide.
 10. An immunogenic reagent according toclaim 9 wherein said further peptide is glutathione-S-transferase (GST).11. A nucleic acid which encodes a polypeptide of an immunogenic reagentaccording to any one of the preceding claims.
 12. An expression vectorcomprising a nucleic acid according to claim
 11. 13. A cell transformedwith a vector according to claim
 12. 14. A method for producing animmunogenic polypeptide which produces an immune response which isprotective against B. anthracis, said method comprising transforming anE. coli host with a nucleic acid which encodes either (a) the protectiveantigen (PA) of Bacillus anthracis or a variant thereof which canproduce a protective immune response, or (b) a protective domain of theprotective antigen (PA) of Bacillus anthracis or a variant thereof whichcan produce a protective immune response, culturing the transformed hostand recovering the polypeptide therefrom, provided that where thepolypeptide is the protective antigen (PA) of Bacillus anthracis avariant thereof which can produce a protective immune response, thepercentage of guanidine and cytosine residues within the said nucleicacid is in excess of 35%.
 15. A method according to claim 14 wherein thesaid nucleic acid encodes the protective antigen (PA) of Bacillusanthracis or a variant thereof which can produce a protective immuneresponse.
 16. A method according to claim 15 wherein the percentage ofguanidine and cytosine residues within the said nucleic acid is inexcess of 45%.
 17. A method according to claim 16 wherein the percentageof guanidine and cytosine residues within the said nucleic acid is from50-52%.
 18. A method according to claim 14 wherein the said nucleic acidencodes a protective domain of the protective antigen (PA) of Bacillusanthracis or a variant thereof which can produce a protective immuneresponse.
 19. A method according to claim 18 wherein the domain isdomain 1 and/or domain 4 of PA of B. anthracis.
 20. A recombinantEscherichia coli cell which has been transformed with a nucleic acidwhich encodes the protective antigen (PA) of Bacillus anthracis or avariant thereof which can produce a protective immune response, andwherein the percentage of guanidine and cytosine residues within thenucleic acid is in excess of 35%.
 21. A recombinant Escherichia colicell according to claim 20 wherein the percentage of guanidine andcytosine residues within the said nucleic acid is in excess of 45%. 22.A recombinant Escherichia coli cell according to claim 21 wherein thepercentage of guanidine and cytosine residues within the said nucleicacid is from 50%-52%.
 23. A recombinant E. coil cell according to claim20 wherein said nucleic acid is of SEQ ID NO 1 as shown in FIG. 2 or amodified form thereof.
 24. A recombinant E. coli cell according to claim23 wherein said nucleic acid is of SEQ ID NO
 1. 25. A recombinantEscherichia coli cell which has been transformed with a nucleic acidwhich encodes a protective domain of the protective antigen (PA) ofBacillus anthracis or a variant thereof which can produce a protectiveimmune response.
 26. A recombinant cell according to claim 25 whereinthe nucleic acid encodes domain 1 or domain 4 of PA of B. anthracis. 27.A method of producing a polypeptide which produces an immune responsewhich is protective against B. anthracis, said method comprisingculturing a cell according to any one of claims 20 to 26 and recoveringthe protective polypeptide from the culture.
 28. An E. colitransformation vector comprising a nucleic acid which encodes theprotective antigen (PA) of Bacillus anthracis or a variant thereof whichcan produce a protective immune response, and wherein the percentage ofguanidine and cytosine residues within the nucleic acid is in excess of35%.
 29. An E. coli transformation vector comprising a nucleic acidwhich encodes a protective domain of the protective antigen (PA) ofBacillus anthracis or a variant thereof which can produce a protectiveimmune response.
 30. A nucleic acid of SEQ ID NO 1 or a modified formthereof which encodes PA or a variant thereof which produces aprotective immune response and which has at least 35% GC content.
 31. Anucleic acid according to claim 30 which is at least 90% identical toSEQ ID NO
 1. 32. A nucleic acid according to claim 31 which comprisesSEQ ID NO
 1. 33. A method of preventing or treating infection by B.anthracis, said method comprising administering to a mammal in needthereof, a sufficient amount of an immunogenic reagent according to anyone of claims 1 to
 10. 34. The use of an immunogenic reagent accordingto any one of claims 1 to 10 in the preparation of a medicament for theprophylaxis or treatment of B. anthracis infection.